Cyclin B1 Promoter Research Articles

Abstract P1019: CycleTrack , A Genetic Method To Trace Cardiomyocyte Renewal In Small And Large Animal Models

To what extent cardiac renewal occurs in adulthood and can be stimulated by therapeutic interventions is still a matter of investigation. In particular, over the last few years, significant effort has been made into awakening the endogenous regenerative potential of the adult mammalian heart after injury using different strategies. However, these studies are significantly hindered by the lack of tools to properly assess cardiomyocyte renewal over time. We have developed a genetic strategy, which we named CycleTrack, allowing a cumulative and accurate estimate of cardiomyocyte divisions in vivo. This method is based on the expression, in cardiomyocytes, of the Cre recombinase under the control of a 312-bp fragment of the Cyclin B2 promoter, which is specifically sensitive to cell proliferation. Replication-activated Cre acts on floxed GFP that is either present in the genome of transgenic mice or is exogenously delivered using Adeno-Associated Viral (AAV) vectors. As a result, cardiomyocytes traversing G2/M become irreversibly labeled. Using CycleTrack, we could monitor cardiomyocyte turnover in several physiological and pathological conditions. These included the measurement of the rate of proliferation of cardiomyocytes after apical resection in neonatal mice, the pro-regenerative effect of AAV9-mediated delivery of miRNA-199a and miRNA-590 after myocardial infarction in adult mice and the effect of pregnancy on myocardial hyperplasia. We also delivered the CycleTrack vectors into pig hearts to visualize cardiomyocyte turnover after myocardial infarction. Considering the large availability of Cre reporter mouse lines and the efficacy of AAVs to transfer genes into cardiomyocytes, we propose CycleTrack as a robust and straightforward tool to trace mitotic events for cardiac regeneration studies in small and large animal models.

Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

BackgroundDespite recent advances in diagnostic and therapeutic approaches for gastric cancer (GC), the survival of patients with advanced GC remains very low. Islet-1 (ISL1) is a LIM-homeodomain transcription factor, which is upregulated and promotes cell proliferation in GC. The exact mechanism by which ISL1 influences GC development is unclear.MethodsCo-immunoprecipitation (co-IP) and glutathione S-transferase (GST)-pulldown assays were employed to evaluate the interaction of ISL1 with CDK1. Western blot and immunohistochemistry analyses were performed to evaluate the ability of CDK1 to phosphorylate ISL1 at Ser 269 in GC cell and tissue specimens. Chromatin immunoprecipitation (ChIP), ChIP re-IP, luciferase reporter, and CCK-8 assays were combined with flow cytometry cell cycle analysis to detect the transactivation potency of ISL1-S269-p and its ability to promote cell proliferation. The self-stability and interaction with CDK1 of ISL1-S269-p were also determined.ResultsISL1 is phosphorylated by CDK1 at serine 269 (S269) in vivo. Phosphorylation of ISL1 by CDK1 on serine 269 strengthened its binding on the cyclin B1 and cyclin B2 promoters and increased its transcriptional activity in GC. Furthermore, CDK1-dependent phosphorylation of ISL1 correlated positively with ISL1 protein self-stability in NIH3T3 cells.ConclusionsISL1-S269-p increased ISL1 transcriptional activity and self-stability while binding to the cyclinB1 and cyclinB2 promoters promotes cell proliferation. ISL1-S269-p is therefore crucial for tumorigenesis and potentially a direct therapeutic target for GC.

Toxoplasma gondii ROP16 kinase silences the cyclin B1 gene promoter by hijacking host cell UHRF1-dependent epigenetic pathways

Toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii, is one of the most common infections in the world due to the lifelong persistence of this parasite in a latent stage. This parasite hijacks host signaling pathways through epigenetic mechanisms which converge on key nuclear proteins. Here, we report a new parasite persistence strategy involving T. gondii rhoptry protein ROP16 secreted early during invasion, which targets the transcription factor UHRF1 (ubiquitin-like containing PHD and RING fingers domain 1), and leads to host cell cycle arrest. This is mediated by DNMT activity and chromatin remodeling at the cyclin B1 gene promoter through recruitment of phosphorylated UHRF1 associated with a repressive multienzymatic protein complex. This leads to deacetylation and methylation of histone H3 surrounding the cyclin B1 promoter to epigenetically silence its transcriptional activity. Moreover, T. gondii infection causes DNA hypermethylation in its host cell, by upregulation of DNMTs. ROP16 is already known to activate and phosphorylate protective immunity transcription factors such as STAT 3/6/5 and modulate host signaling pathways in a strain-dependent manner. Like in the case of STAT6, the strain-dependent effects of ROP16 on UHRF1 are dependent on a single amino-acid polymorphism in ROP16. This study demonstrates that Toxoplasma hijacks a new epigenetic initiator, UHRF1, through an early event initiated by the ROP16 parasite kinase.

E2F is required for STAT3-mediated upregulation of cyclin B1 and Cdc2 expressions and contributes to G2-M phase transition.

Activation of transcription factor STAT3 is involved in cell proliferation, differentiation, and cell survival. Constitutive activation of STAT3 pathway has been associated with the oncogenesis of various types of cancers. It has been reported that STAT3 plays a key role in the G1 to S phase cell cycle transition induced by the cytokine receptor subunit gp130, through the upregulation of cyclins D1, D2, D3, A, and Cdc25A and the concomitant downregulation of p21 and p27. However, its role in mediating G2-M phase transition has not been studied. The cyclin B1/Cdc2 complex is widely accepted as the trigger of mitosis in all organisms and is believed to be necessary for progression through S phase and keep active during the G2-M transition and progression. In the present study, we found that activation of STAT3 stimulates cyclin B1 and Cdc2 expressions. Deletion and site-directed mutations on cyclin B1 and Cdc2 promoters indicated that E2F element mediates the upregulation of these two promoters in a STAT3-dependent manner. The findings reported here demonstrated that STAT3 participates in modulating G2-M phase checkpoint by regulating gene expressions of cyclin B1 and Cdc2 via E2F.

Evaluation of the plant growth-promoting activity of Pseudomonas nitroreducens in Arabidopsis thaliana and Lactuca sativa.

Pseudomonas nitroreducens: strain IHB B 13561 (PnIHB) enhances the growth of Arabidopsis thaliana and Lactuca sativa via the stimulation of cell development and nitrate absorption. Plant growth-promoting rhizobacteria (PGPR) enhance plant development through various mechanisms; they improve the uptake of soil resources by plants to greatly promote plant growth. Here, we used Arabidopsis thaliana seedlings and Lactuca sativa to screen the growth enhancement activities of a purified PGPR, Pseudomonas nitroreducens strain IHB B 13561 (PnIHB). When cocultivated with PnIHB, both species of plants exhibited notably improved growth, particularly in regard to biomass. Quantitative reverse transcription polymerase chain reaction analysis indicated high expression levels of the nitrate transporter genes, especially NRT2.1, which plays a major role in the high-affinity nitrate transport system in roots. Moreover, enhanced activity of the cyclin-B1 promoter was observed when wild-type 'Columbia-0' Arabidopsis seedlings were exposed to PnIHB, whereas upregulation of cyclin-B also occurred in the inoculated lettuce seedlings. Overall, these results suggest that PnIHB improves A. thaliana and L. sativa growth via specific pathways involved in the promotion of cell development and enhancement of nitrate uptake.

Real-time imaging of actin filaments in the zebrafish oocyte and embryo.

Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis.

P53 can repress transcription of cell cycle genes through a p21WAF1/CIP1-dependent switch from MMB to DREAM protein complex binding at CHR promoter elements

The tumor suppressor p53 plays an important role in cell cycle arrest by downregulating transcription. Many genes repressed by p53 code for proteins with functions in G₂/M. A large portion of these genes is controlled by cell cycle-dependent elements (CDE) and cell cycle genes homology regions (CHR) in their promoters. Cyclin B2 is an example of such a gene, with a function at the transition from G₂ to mitosis. We find that p53-dependent downregulation of cyclin B2 promoter activity is dependent on an intact CHR element. In the presence of high levels of p53 or p21WAF1/CIP1, protein binding to the CHR switches from MMB to DREAM complex by shifting MuvB core-associated proteins from B-Myb to E2F4/DP1/p130. The results suggest a model for p53-dependent transcriptional repression by which p53 directly activates p21WAF1/CIP1. The inhibitor then prevents further phosphorylation of p130 by cyclin-dependent kinases. The presence of hypophosphorylated pocket proteins shifts the equilibrium for complex formation from MMB to DREAM. In the case of promoters that do not hold CDE or E2F elements, binding of DREAM and MMB solely relies on a CHR site. Thus, p53 can repress target genes indirectly through CHR elements.

Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching

The malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor (AR) in the surrounding stroma. However, the function and mechanisms of AR signaling in prostate cancer (PCa) stroma remain elusive. Here we report, by using proteomics pathway array analysis (PPAA), that androgen and its receptor inhibit the proliferation of prostate stromal cells through transcriptional suppression of cyclin B1, and we confirmed our findings at mRNA and protein levels using AR-negative or -positive primary prostate stromal cells. Furthermore, AR showed a negative correlation with cyclin B1 expression in stroma of human PCa samples in vivo. Mechanistically, we identify cyclin B1 as a bona fide AR target gene in prostate stromal cells. The negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to the cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR-negative stromal cells or AR-positive stromal cells when androgens are depleted. Upon stimulation with androgen in AR-positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to the recruitment of the silencing mediator for retinoid and thyroid hormone receptor (SMRT)/histone deacetylase 3 (HDAC3) corepressor complex and repression of cyclin B1 at the chromatin level. The switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as the cytoplasmic-to-nuclear translocation of E2F4. Thus, we identified a novel mechanism for E2F factors in the regulation of cell cycle gene expression and cell cycle progression under the control of AR signaling.

Abstract SY09-02: Molecular mechanisms in prostate cancer in African Americans

Abstract African American men have long been known to have the highest rates of prostate cancer (PCa) in the world. In addition, age adjusted mortality rates from PCa patients are two times worse among African Americans (AA) than Caucasians (CA). The reasons for the disparity in PCa risk, incidence, clinical progression, and disease-specific death are still controversial. In addition to socioeconomic and environmental factors, such as diet, access to care, and screenings, a biologically more aggressive cancer is proposed as one important explanation for the racial disparity observed in AA men. Androgens and the androgen receptor pathway are well established key regulators of aggressive PCa behavior. Using a novel proteomic pathway analysis comparing the distinct signal pathway and network proteins different between AA and CA PCa, we identified a number of markers including stem/progenitor cell and EMT related proteins that are overexpressed in PCa among AA. Our ongoing study indicates a novel link among Notch, WNT and androgen receptor (AR) pathways. In contrast to increase in epithelial AR, our data revealed a decrease in the expression of the stromal AR in AA PCa compared to CA PCa. Androgen and its receptor inhibit proliferation of prostate stromal cells through transcriptional suppression of cyclin B1. Mechanistically, the negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR negative stromal cells or AR positive stromal cells under androgen depleted condition. Upon stimulation with androgen in AR positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to recruitment of the SMRT/HDAC3 co-repressor complex and repression of cyclin B1. Furthermore, the switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as cytoplasm to nucleus translocation of E2F4. In summary, our study revealed an association between distinct epithelial and stromal AR expression and PCa racial disparity as well as the molecular mechanism involved in regulating AR expression in epithelial and stromal prostate cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY09-02. doi:1538-7445.AM2012-SY09-02

Regulation of the Potential Marker for Intestinal Cells, Bmi1, by β-Catenin and the Zinc Finger Protein KLF4

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.

The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G0/G1. It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G0. Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G0/G1, but also for activation in S, G2 and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle.

Hydrogen peroxide induces Sp1 methylation and thereby suppresses cyclin B1 via recruitment of Suv39H1 and HDAC1 in cancer cells

Sp1 is an important transcription factor for a number of genes that regulate cell growth and survival. Sp1 is an anchor protein that recruits other factors to regulate its target genes positively or negatively, but the mechanism of its functional switch by which positive or negative coregulators are recruited is not clear. In this study, we found that Sp1 could be methylated and that methylation was maintained by treatment with pargyline, a lysine-specific demethylase 1 (LSD1) inhibitor or knock LSD1 down directly. Hydrogen peroxide treatment increased the methylation of Sp1 and repressed Sp1 transcriptional activity. Investigation of the mechanism by which methylation decreased Sp1 activity found that methylation of Sp1 increased the recruitment of Su(var) 3–9 homologue 1(Suv39H1) and histone deacetylase 1 (HDAC1) to the cyclin B1 promoter, resulting in deacetylation and methylation of histone H3 and subsequent downregulation of cyclin B1. Finally, downregulation of cyclin B1 led to cell cycle arrest at the G2 phase. These results show that methylation of Sp1 causes it to act as a negative regulator by recruiting Suv39H1 and HDAC1 to induce chromatin remodeling. This finding that methylation acts as a functional switch provides new insight into the modulation of Sp1 transcriptional activity.

Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum.

BackgroundPlants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood.Methodology/Principal FindingsWe developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation.ConclusionsWe provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism.

Phosphorylation of HOX11/TLX1 on Threonine-247 during mitosis modulates expression of cyclin B1

BackgroundThe HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11.ResultsCell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis.ConclusionsOur results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that phosphorylation functions to regulate the expression of HOX11 target genes involved in the control of the mitotic spindle checkpoint.

The CCAAT/Enhancer Binding Protein β Is a Critical Regulator of Steroid-Induced Mitotic Expansion of Uterine Stromal Cells during Decidualization

During early pregnancy, the concerted actions of the maternal steroid hormones, estrogen and progesterone, promote a unique process known as decidualization, which involves extensive proliferation and differentiation of uterine stromal cells. The molecular pathways underlying this hormonally induced cellular transformation, an essential prerequisite for embryo implantation, remain poorly understood. We previously identified CCAAT/enhancer binding protein beta (C/EBPbeta) as a target of steroid regulation in the uterus. Uteri of mice lacking C/EBPbeta failed to undergo decidualization. In the present study, analyses of C/EBPbeta-null uteri indicated that loss of this factor leads to a block in stromal cell proliferation in response to a decidual stimulation. The mutant stromal cells entered S phase of the cell cycle and completed DNA synthesis but were unable to execute mitosis. Further analysis revealed that C/EBPbeta facilitates the transition of these cells into mitosis by binding directly to the cyclin B2 promoter to regulate its expression. The expression of cdc25C, a phosphatase that maintains the active state of the cyclin B-cyclin-dependent kinase complex during mitosis, is also strongly suppressed in C/EBPbeta-null stromal cells. Furthermore, the expression of the tumor suppressor p53 and the cell cycle inhibitors p21 and p27 was markedly elevated in C/EBPbeta-null stromal cells before the mitotic phase, uncovering additional mechanisms by which C/EBPbeta controls G2 to M transition. Collectively, these results revealed that C/EBPbeta mediates the effects of steroid hormones during decidualization by modulating the expression of multiple key cell cycle regulatory factors that control the G2 to M transition of the proliferating uterine stromal cells.

Abstract 1070: Circadian transcriptional factor Period 2 modulates cyclin B1 expression

Abstract The proper timing of cell division is a major factor contributing to the regulation of normal growth and emerges as a fundamental process in the development of most cancers. The existence in most, if not all, living organisms of many behavioral and physiological events that occur at well-defined and controlled times argues for the presence of time-keeping systems that allow them to adapt to cyclic changes in environmental conditions. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression. In the present study, we use cDNA microarray analysis to identify Per2 target genes following siRNA knock-down expression of Per2 in mouse hepatocytes AML12 cells. We identified cyclin B1, a key G2/M regulator of the cell cycle, as a downstream target gene that is negatively regulated by Per2 under normal conditions. Quantitative real-time PCR confirmed our observations by showing cyclin B1 expression is enhanced under Per2 knock-down condition. Accordingly, Bmal1 expression, a target gene of Per2, was specifically augmented under Per2 over-expression conditions. Further experiments focused in characterizing the regulation of the human cyclin B1 promoter by Per2 using luciferase reporter assays. Results show cyclin B1 reporter activity is enhanced in siPer2 transfected cells compared to control scrambled siRNA. Promoter analysis defined the region comprising −484 to −284 of human cyclin B1 promoter containing response elements for gene trans-activation while the region between −284-1 is mainly responsible for Per2 regulation. Our results show the canonical E-box between −284-1 is not required for Per2 regulation of cyclin B1, suggesting non-canonical E-boxes might be directly responsible for Per2-mediated expression. FACS analysis revealed that siPer2 treated cells exhibit a reduced G2/M phase with correspondingly higher cyclin B1 protein levels and reduced Tyr-15-cdc2 phosphorylation compared to scrambled siRNA treated cells. Our previous results show Per2 stability is directly controlled by the endogenous levels of heme whose endogenous levels oscillate in a 24 h cycle. Here, we evaluated whether cyclin B1 expression is altered in heme-treated cells. In contrast to its up-regulation in siPer2 treated cells, luciferase reporter assay showed a decrease in cyclin B1 promoter activity in response to heme treatment. These results may reflect a complex pattern of regulation in which Per2 acts as a sensor molecule to monitor changes in both environmental and cellular conditions that will impact cell progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1070.

PPARα ligands cause lymphocyte depletion and cell cycle block and this is associated with augmented TRB3 and reduced Cyclin B1 expression

PPARα ligands are medications used clinically to prevent cardiovascular events, however studies have shown that these agents are also anti-inflammatory. Our previous studies have shown that PPARα ligands induce lymphocyte depletion. PPARα ligands also potently upregulate TRB3, a protein that has been associated with cell cycle arrest. Therefore the following studies were undertaken to determine the mechanisms associated with lymphocyte depletion. Our studies demonstrate that WY14,643, a PPARα ligand, decreases the amount of lymphocytes recovered after stimulation and reduces cellular divisions. Cells treated with WY14,643 also accumulate in the G2/S phase of the cell cycle. TRB3 has been shown to inhibit the phosphorylation of AKT/Protein Kinase B, and reduced activation of AKT has been associated with decreased cellular divisions and survival. However in lymphocytes, TRB3 did not reduce the phosphorylation of AKT, and WY14,643 treatment was associated with enhanced activation of AKT. Drosophila tribbles (TRB3 homolog) causes G2 arrest by decreasing the expression of a Cdc25c homolog. Lymphocytes stimulated and treated with WY14,643 have reduced expression of Cdc25c, however this is not associated with enhanced expression of phosphorylated-Cdc2 which induces G2 arrest. Instead we observed that WY14,643 consistently reduces the protein and mRNA expression of Cyclin B1. Moreover, TRB3 inhibits activation of a Cyclin B1 promoter construct. In summary, we propose that PPARα ligands may reduce cellular number by augmenting TRB3 expression, which in turn induces cell cycle arrest by reducing the expression of Cyclin B1. Reduced cellular divisions and cell cycle arrest may be responsible for some of the immunomodulatory effects of these agents that have been consistently observed in human trials.

Downregulation of p21‐activated kinase‐1 inhibits the growth of gastric cancer cells involving cyclin B1

p21-Activated kinase 1 (Pak1), a serine/threonine kinase, has been implicated in cytoskeletal remodelling, cell motility, apoptosis and transformation. However, the role of Pak1 in gastric cancer remains unclear. In this study, we detected Pak1 expression in gastric cancer tissues from 40 patients by western blot. Overexpression of Pak1 was associated with progression, metastasis and prognosis of gastric cancer. In addition, we found that knockdown of Pak1 expression significantly inhibited anchorage-dependent and anchorage-independent growth in gastric cancer cells, and markedly inhibited gastric cancer cell xenograft tumor growth. In further study, data showed that Pak1 could regulate the expression of cyclin B1 at the mRNA and protein levels, and impact the subcellular distribution and the promoter activity of cyclin B1. Results from deletion and mutant analysis supplied a new NF-kappaB binding sites at position -321 of cyclin B1 promoter, and indicated that Pak1 regulated the transcription of cyclin B1 in gastric cancer through NF-kappaB. In conclusion, Pak1 may be a potential prognostic marker and therapeutic target in gastric cancer.

Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells.

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post-translational modifications control diverse chromatin functions. Here, we report our findings from a large-scale screen for DNA-damage-responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA-damage-induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell-cycle re-positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl-transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.

Cdk5-mediated Phosphorylation of c-Myc on Ser-62 Is Essential in Transcriptional Activation of Cyclin B1 by Cyclin G1

It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G2 arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.